1. Field of the Invention
The present invention relates to the field of blood and blood products. More specifically, it relates to platelets and platelet compositions, particularly those containing freeze-dried platelets or rehydrated freeze-dried platelets, that are useful for therapeutic, diagnostic, and research purposes.
2. Description of Related Art
Blood is a complex mixture of numerous components. In general, blood can be described as comprising four main parts: red blood cells, white blood cells, platelets, and plasma. The first three are cellular or cell-like components, whereas the fourth (plasma) is a liquid component comprising a wide and variable mixture of salts, proteins, and other factors necessary for numerous bodily functions. The components of blood can be separated from each other by centrifugation. Typically, centrifugation results in a large volume/mass of the dense red blood cells migrating to the bottom of the centrifuge tube. Above the red blood cells, one will find a relatively thin layer of white blood cells and platelets, which is known as the “buffy coat”, due to its whitish grey color. Above the buffy coat is the liquid plasma fraction.
Red blood cells, which are also commonly referred to as erythrocytes, are responsible for carrying oxygen from the lungs to cells for use in cellular processes, and waste carbon dioxide from cells to the lungs for excretion. Red blood cells do not have a nucleus, and are thus short lived cellular components of blood that are constantly being replaced in healthy individuals. The percentage of blood volume composed of red blood cells is called the hematocrit, and this number is often used to indicate the presence of one or more diseases or disorders of or affecting the blood system. Normal hematocrit values are between 37% and 47% for females, and 40% and 54% for males. Red blood cells are routinely transfused into patients in need of them, such as those with chronic anemia or who have sustained an injury or trauma or who have undergone surgery, which resulted in blood loss. In addition, red blood cells are often used to treat anemia caused by any number of diseases or disorders.
White blood cells, which are also commonly referred to as leukocytes, are nucleated cells that are responsible for protecting the body from damage caused by foreign substances. As a general rule, white blood cells function to combat pathogenic organisms, such as bacteria, fungi, and viruses, or substances that might be detrimental to the body, such as protein toxins. However, in certain individuals, white blood cells mount a protective response against apparently harmless substances, such as pollen, resulting in allergic reactions. Indeed, in some cases, white blood cells inappropriately react against a body's own cells or proteins, resulting in autoimmune diseases and destruction of body tissues, which can, in certain circumstances, be fatal. Among other things, purified white blood cells have found use in treating patients who are unresponsive to antibiotic therapy.
Platelets, which are also commonly referred to as thrombocytes, are small, irregularly-shaped megakaryocyte-derived components of blood that are formed in the bone marrow and are involved in the clotting process, and thus aid in protecting the body from excessive blood loss due not only to trauma or injury, but to normal physiological activity as well. Indeed, platelets are crucial in normal hemostasis, providing the first line of defense against blood escaping from injured blood vessels. Platelets generally function by adhering to the lining of blood vessels and interacting with components of the clotting system that are present in plasma or are released by other cellular components of the blood. Purified platelets have found use in treating patients with abnormal platelet function (thrombasthenia) and low platelet count (thrombocytopenia). Concentrated platelets are often used to control bleeding after injury or during acquired platelet function defects, for example those occurring during bypass surgery. The normal circulating platelet count is between 150,000 and 450,000 per microliter (ul) of blood.
When bleeding from a wound suddenly occurs, the platelets gather at the wound and attempt to block the blood flow by forming a clot. There are two general mechanisms to clot formation. In one mechanism, a clot begins to form when the blood is exposed to air. The platelets sense the presence of air and react with fibrinogen to begin forming fibrin. The resulting fibrin forms a web-like mesh that traps blood cells within it. In the other general mechanism, damaged blood vessels release a chemical signal that increases the stickiness of platelets in the area of the injury. The sticky platelets adhere to the damaged area and gradually form a platelet plug. At the same time, the platelets release a series of chemical signals that prompt other factors in the blood to reinforce the platelet plug. Between the platelet and its reinforcements, a sturdy clot is created that acts as a patch while the damaged area heals.
Platelets, in the form of platelet gels, have been used extensively to accelerate wound healing and, in conjunction with autologous fibrin glue, autologous platelet gel has been shown to improve perioperative hemostasis and reduce blood transfusion needs in surgery to replace the ascending aorta (Christenson and Kalangos, 2004). Costasis Surgical Hemostat (Costatis®). A combination of bovine thrombin, bovine collagen, and plasma as the source of fibrinogen and platelets has been shown to work well in the in vivo bleeding rabbit kidney and spleen model (Prior et al., 1999). Nevertheless, other studies have shown that platelet gel, when used alone, is not an effective hemostasis agent (Wajon et al., 2001). Despite of the contradicting findings regarding platelets and their role as hemostasis agents, there is little doubt about the pro-coagulant nature of platelet microparticles; these essential components, often overlooked, are increasingly being recognized as active participants in the in vitro and in vivo clotting process (Nieuwland et al., 1997). When platelets are stimulated with a combination of physiological agonists, such as thrombin and collagen, they release large quantities of microparticles (Sims et al., 1988; Tans et al., 1991). The activated platelets and microparticles express an aminophospholipid, which provides a procoagulant surface to support the formation of activated clotting enzymes in the intrinsic, extrinsic, and common pathways (Rosing et al., 1985).
Compared with activated platelets, microparticles contain a higher density of high-affinity binding sites for activated factor IX (IXa) (Hoffman et al., 1992) and factor Va (Sims et al., 1988). They have a continuous expression of high-affinity binding sites for factor VIII (Gilbert et al., 1991) and support both factor Xa activity (Gilbert et al., 1991; Holme et al., 1995) and prothrombinase activity (Sims et al., 1989).
Aside from the fact that platelet microparticles are important components in the hemostatic response, platelets, in the form of platelet gels, have been used in surgical wound healing applications as well as to treat difficult to heal wounds (Mazzucco et al., 2004). Moreover, the use of platelets in the form of platelet rich plasma has expanded into novel applications, such as bio-tissue engineering or autologous and allogenic tissue grafts, as well as osseous bone integration and soft tissue regeneration (Oikarinen et al., 2003). This is because platelets contain a number of important growth factors within their alpha granules that contribute to the process of hemostasis and wound healing. Studies have found that growth factors, such as platelet derived wound healing factors (PDWHF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), and insulin growth factors (IGF), among others, are important in different stages of the wound-healing cascade and greatly influence mitogenic and cellular differentiation activities (Pierce et al., 1989; Steed, 1997).
These findings have lead to the development of strategies for growth factor replacement. For example, Regranex®, a recombinant human PDGF in a carrier gel, is used to treat diabetic wounds, while others, such as TGF, are currently being tested for FDA approval. Nevertheless, a single growth factor applied into a wound is not as effective as multiple growth factors. This is not surprising since wound healing is a complex integration of cascades that requires multiple growth factors for different stimulatory and inhibitory functions at different phases within the process.
The liquid portion of the blood, which is commonly referred to as plasma, is a complex solution containing various proteins and salts. In general terms, plasma is the substance that remains when red blood cells, white blood cells, and platelets are removed from blood. Due to the presence of numerous proteins in high concentrations, plasma is a straw colored liquid that is unstable at room temperature (i.e., plasma must be stored well below room temperature to protect the proteins present in it from losing activity). The major protein constituents of plasma are: albumin; fibrinogen; antibodies; and numerous proteins necessary for clotting and hemostasis. As can be seen from this brief listing of plasma proteins, plasma serves a variety of functions, from maintaining a satisfactory blood pressure and providing volume to supplying critical proteins for blood clotting and immunity. For example, gamma globulin isolated from plasma can be used to treat patients in need of an antitoxin, and the presence of certain antibodies can be assayed to indicate whether a patient is infected with a certain virus or bacteria. In addition, clotting factor VIII, which can be isolated from plasma, is often used in the treatment of classical congenital hemophilia.
The major functions of blood are to transport oxygen and carbon dioxide and to enable immune system components to quickly and effectively reach all parts of the body to fight off invading microbes. However, because blood is a fluid and needs to be not only retained within the body, but restricted to specific areas of the body (such as blood vessels or other parts of the circulatory system), an important function of blood is to monitor its own distribution within the body, and repair damage that permits the blood to escape from the body or specific areas within the body where it should be retained. The process of monitoring and maintaining blood distribution within its normal boundaries is a balance of the physiological processes that, on the one hand, prevent excessive bleeding after vessel injury (through formation of clots), while on the other hand, maintain a normal blood circulation by keeping the blood in an uncoagulated (i.e., unclotted) fluid state. These seemingly competing processes are part of a complex system that has many control points and feedback loops.
The main process for maintenance of proper blood flow and containment is called hemostasis, which is the process of formation and ultimate degradation of blood clots and the repair of injured tissue. Hemostasis is comprised of four main events: vascular constriction; aggregation of platelets at the site of injury, mediated by fibrinogen, and activation of the platelets by thrombin; creation of a clot (also referred to as a thrombus or fibrin mesh) by platelets and a complex interaction of numerous clotting factors; and, finally, degradation of the clot and repair of the injured tissue.
Blood clotting is a complicated process: if the clot formation is unchecked, the vessel will become occluded; if the clot is not sturdy, excessive blood loss will occur. Therefore, a delicate balance must be maintained for normal hemostasis. In situation where normal hemostasis is unbalanced, clot formation may be compromised. Such an abnormality could be acquired due to ingestion of aspirin or caused by immune dysfunction. The abnormality could also be congenital, such as through genetic diseases and clotting factor defects. For example, defects in the process of hemostasis that lead to bleeding disorders have been identified, and most of such defects are in the enzymes involved in the cascade of activities required for clotting, in platelet activation and function, or in contact activation. Included among these disorders are vWD and hemophilia. Other diseases or disorders of the blood clotting system are a result (i.e., side effect) of treatments for other diseases or disorders. Treatments for such diseases and disorders typically involve reducing the dose of the drug causing the side effect, or discontinuing treatment with the drug.
Blood clotting relies on a complex cascade of enzymatic activities that are tightly controlled through numerous feedback loops and control points. Clotting begins when platelets adhere to the cut wall of an injured blood vessel or other lesion site. In doing so, platelets adhere to collagen that is present on cells at the site of injury, a process that is mediated by a clotting factor known as von Willebrand factor (vWF). vWF is a complex protein that is produced in megakaryocytes and endothelial cells, and stored in platelets or in certain connective tissues. It is often found complexed with Factor VIII, and is known to be necessary for stabilization of Factor VIII in plasma. Defects in quantity and function of vWF are typically genetic in nature, and result in a disease known as von Willebrand disease (vWD).
Adhesion of platelets to the site of injury is mediated by vWf binding to collagen in the subendothelium. Fibrinogen, which exists in plasma as a soluble protein, can bridge activated platelets together in a process termed aggregation or cohesion. Fibrinogen is converted to insoluble strands of fibrin by the enzyme thrombin (which is activated by activated Factor X (Factor Xa)), which also is a potent platelet activating agent. The fibrin, which spontaneously polymerizes into filaments, binds to surface proteins or phospholipids on the platelets to ensnare the platelets in a mesh. The fibrin filaments are then cross-linked through the activity of Factor XIIIa, which is formed from Factor XIII by thrombin. The fibrin-platelet mesh that forms is referred to as a fibrin mesh, thrombus, or clot.
Factor X can be activated by either of two pathways, termed the extrinsic and intrinsic pathways. The intrinsic pathway involves a series of enzymatic reactions that activates various proteases. The process begins with binding of Factor XII to a negatively charged surface, presumably supplied by components of the subendothelium, and activation of Factor XII to Factor XIIa by Kallikrein in a reaction mediated by High Molecular Weight Kininogen (HMWK). Factor XIIa then converts Factor XI to Factor XIa (plasma thromboplastin antecedent). In the presence of calcium ions, Factor XIa converts Factor IX to its activated form, Factor IXa. Factor IXa combines with the non-enzyme protein Factor VIII (antihemophilic globulin or AHG), and in the presence of calcium ions and cell derived phospholipids, activates circulating Factor X to form Factor Xa.
In the extrinsic pathway, which is widely regarded as the primary physiological pathway for initiation of coagulation, the activated form of Factor VII, Factor VIIa, associates with Factor m (tissue thromboplastin), commonly referred to as tissue factor (TF). In the presence of calcium ions, the Factor VIIa/TF complex activates circulating Factor X to form Factor Xa. Factor Xa can also be formed from the action of Factor VII through Factors IX and XI. In this scenario, Factors IX and X can be activated by the combined activities of TF and Factor VIIa. The Factor VIIa/TF complex is recognized as the most potent trigger of the clotting cascade. As discussed above, Factor IXa, in the presence of calcium, phospholipids on the surface of platelets, and Factor VIIIa, activates Factor X to Factor Xa, which then converts prothrombin to thrombin. Thrombin converts soluble fibrinogen to insoluble fibrin fibers to create a mesh.
Thus, Factor X is activated by either the intrinsic or extrinsic activation pathway. Factor Xa, with activated Factor V and in the presence of calcium ions and phospholipids present on the surface of platelets, activates prothrombin to thrombin, which forms fibrin from fibrinogen, leading to formation of a clot. The coagulation of blood is a complex process that involves interaction of a number of components, including fibrinogen, thrombin, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, and Factor XII. The loss of one of these components invariably leads to a clinical presentation of a blood disorder, which can be life-threatening for some patients.
Defects in the process of hemostasis that lead to bleeding disorders have been identified, and most of such defects are in the enzymes involved in the cascade of activities required for clotting, in platelet activation and function, or in contact activation. Included among these disorders are vWD and hemophilia. Various treatments for these two disorders are known, most of which rely on supplying one or more of the clotting factors mentioned above.
Congenital Hemophilia is classified in three different groups: classical hemophilia or Hemophilia A (FVIII deficiency); Christmas disease or Hemophilia B (FIX deficiency); and Hemophilia C (FXI deficiency). Hemophilia is recognized as a disorder in which bleeding is not stopped within the normal amount of time. That is, hemophiliacs do not bleed more profusely or more quickly, they bleed for a longer period of time. Approximately 20,000 Americans have hemophilia. The vast majority of cases are either Hemophilia A or B, with Hemophilia A accounting for about 80 percent of all hemophilia cases. Hemophilia C is rare, occurring in approximately one in 100,000 Americans.
Hemophilia A is an X-linked disorder resulting from a deficiency of Factor VIII, and is defined as the absence or less than normal levels of Coagulation Factor VIII. Hemophilia A arises from a variety of mutations in the gene for Factor VIII. Current treatment of Hemophilia A involves infusion of Factor VIII concentrates or concentrates of complexes of Factor VIII and vWF prepared either from human plasma or by recombinant DNA technology. In contrast to Hemophilia A, Hemophilia B results from deficiencies in Factor IX. Current treatment of Hemophilia B involves infusion of plasma-derived or recombinant Factor IX concentrates. Finally, Hemophilia C results from deficiencies in Factor XI.
Due to continuing concerns about the safety of the public blood supply and emerging diseases that are transmissible through blood and blood products, the source of choice for Factor VIII has become a recombinantly produced form. Recombinant Factor IX has been approved for human use (Benefix®, Genetics Institute) and will likely become the source of choice. In addition, gene therapy has been proposed as a treatment or cure for hemophilia. However, to date, transgenic approaches for the treatment of hemophiliacs have not resulted in long term stable expression of coagulation factors, have encountered unanticipated mortality issues, and still may result in inhibitor production in recipients (discussed below).
Treatment of hemophilia with concentrates of Factor VIII causes, in about 15% to about 30% of patients with Hemophilia A, and in about 3% of patients with Hemophilia B, production of antibodies against the introduced Factor VIII or IX, respectively. Although recombinant Factor VIII appears to cause this response in only about 5% of patients with Hemophilia A, it is still a significant problem in the treatment of this disorder. This process and the resulting disorder are referred to as “Hemophilia with Inhibitors”, often described as the induction of antibodies to transfused proteins normally used to treat a missing clotting factor. In contrast, Acquired Hemophilia is the development of inhibitors in persons containing normal levels of coagulation proteins. Acquired Hemophilia is therefore a pseudo-autoimmune disease, and can occur in otherwise normal non-hemophilia individuals who are treated with products containing factors involved in clotting. In general, the antibodies that are generated react with the Factor VIII that is administered, and result in inhibition of the Factor VIII activity, thus rendering the treatment useless in patients lacking endogenous Factor VIII, and ironically rendering the treatment harmful in patients who originally possessed a low, but insufficient, level or activity of endogenous Factor VIII.
Numerous ways of avoiding Acquired Hemophilia and Hemophilia with Inhibitors have been proposed and implemented. For example, rather than treating with exogenous Factor VIII, another strategy to treat Hemophilia A is to administer exogenous Factor VIIa, thus eliminating the need for Factor VIII for hemostasis. Likewise, treating with excessive amounts of Factor VIII and with anti-idiotype anti-Factor VIII antibodies have been tested. Other approaches include using FEIBA bypassing agents, Prothrombin Complex Concentrates, Recombinant Factor VIIa, Porcine Factor VIII, infusion of high dose intravenous Immunoglobulin, Immune Tolerance Therapy (ITT), and plasmapheresis either with or without Protein A adsorption to remove the inhibiting antibodies.
Furthermore, treatment with purified recombinant Factor VIIa has become common. For example, dosages of 10 to 15 ug/kg, and even as high as 150 ug/kg, a range that can provide a circulating level of Factor VIIa of about 0.2 to 2.0 ug/ml blood, of Factor VIIa have been found to be safe and effective in some Hemophilia A patients with inhibitors. These doses are quite high compared to the normal estimated concentration of Factor VIIa, which is about 0.005 ug/ml blood. Although these methods have seen success, none of the current methods are completely effective, and all are quite expensive. Furthermore, at least 5-10% of patients receiving recombinant FVIIa therapy fail to achieve hemostasis.
In addition, Type III or severe von Willebrand Disease often presents clinically as Hemophilia A. Factor VIII is normally transported and protected from plasma proteases by vWf. In the absence of circulating vWf, the endogenous Factor VIII is rapidly degraded and cleared from circulation, resulting in symptoms of Hemophilia A. Treatments for vWD vary depending upon the nature and severity of the disease. Treatments include DDAVP therapy, either by injection or through the nasal passage. The DDAVP therapy acts by releasing endothelial cell vWf to the circulation. Treatments also include plasma cryoprecipitate, which provides a concentrated form of vWf and other clotting factors.
Traditional treatment of hemophilia typically occurs only after bleeding symptoms are recognized. More recent treatment regimens have been developed in which periodic prophylactic infusion of missing clotting factors is performed, regardless of bleeding status at the time. This approach maintains the factor level high enough that bleeding, joint destruction, and life-threatening hemorrhage are minimized and almost entirely avoided. While highly effective, this therapy regimen is quite expensive.
Platelet functionality is another critical component of blood clots. Dysfunctional platelets may lead to abnormal hemorrhage, such as bleeding or thrombosis. Thus platelet function assays are an integral part of the diagnosis and monitoring of blood related diseases. For example, acquired platelet defects, such as ingestion of aspirin, cardiac disease, renal disease, or congenital platelet defects such as Bernard-Soulier syndrome, Glanzmann's thrombasthenia and storage pool disease, to name a few, can influence the normal hemostatic function of the platelets. To assess the platelet function, at the very minimal, a complete blood count with a peripheral blood smear will provide some basic information. Other tests, such as bleeding time, platelet function tests using an aggregometer to assess the aggregation of platelets to a panel of platelet agonists performed on whole blood or platelet rich plasma will classify the defect. However, such analyses, although accurate, are not highly sensitive, and can fail to detect slight perturbances in normal clotting function at early stages of a disorder. Likewise, determination of the precise point of failure of the blood clotting cascade may require numerous assays using freshly drawn blood.
Although it is known that platelets are involved in the clotting process and are the source of at least one clotting factor, to date, there is no disclosure of the use of resting, activated, fixed, frozen, or lyophilized platelets, or any combination of these, for the treatment of Acquired or Congenital Hemophilia or for treatment of persons with bleeding disorders who have normal platelet counts and platelet functions. Kirby & Gregoriadis (1984) prepared liposomes containing Factor VIII in an attempt at oral treatment of hemophilia. Later, Giles et al. (1988) showed a combination of Factor Xa and phosphatidylcholine-phosphatidylserine vesicles bypassed Factor VIII in vivo, while Hong & Giles (1992) demonstrated the normalization of the hemostatic plugs of dogs with Hemophilia A (Factor VIII deficiency) following the infusion of a combination of Factor Xa and phosphatidylcholine-phosphatidylserine vesicles. More recently, Yarovoi et al. (2003), using a transgenic approach, demonstrated that Factor VIII ectopically expressed in platelets showed efficacy in Hemophilia A treatment in a mouse model. Further, Hrachovinova et al. (2003) showed that the interaction of P-selectin and PSGL1 generates leukocyte-derived micro particles that correct hemostasis in a mouse model of Hemophilia A. However, none of these researchers used or proposed using normal platelets or platelet derivatives to treat hemophilia.
Typically, detection of a blood clotting disease or disorder involves analyzing the patient's blood for platelet counts, various markers involved in blood clotting, and clot-forming ability. The coagulation assays measured the activated clotting time (ACT), the prothrombin time (PT), the plasma thrombin time (PTT), and the activated partial thromboplatin time (APTT) are used to evaluate the intrinsic and extrinsic coagulation pathways. These assays are generally performed in the laboratory and analysis often requires multiple samples of blood to be drawn from the patients. Moreover, these assays are potentially unreliable as they are end-point tests in which results are based on the time of clot formation in vitro. Another limitation relates to the fact that exogenous reagents, such as kaolin, thrombin, calcium, etc. must be added thus, the results are based on an artificial system, and do not necessarily reflect the patent's thrombotic potential.
As discussed above, a critical function of the blood clotting system is to stop blood loss from injured tissues, such as tissues that have been damaged by wounds, surgery, or other trauma. However, sometimes the wound or trauma is so great that the blood system of the injured person is unable to rapidly and effectively stop all of the bleeding. Furthermore, while the clotting function is provided satisfactorily in most persons, in some persons the clotting system is impaired such that adequate clotting is not provided and extensive, sometimes deadly bleeding occurs as a result of injury or trauma. Thus, there are often times where a person is in need of additional platelets to provide the clotting function that is missing or inadequate.
In addition to their use “as is” to supply blood clotting functions to persons in need, platelets are studied extensively in the laboratory to characterize their properties and understand their precise role in the blood clotting cascade. Research on platelets provides information on blood clotting factors that are provided by the platelets, factors that interact with the platelets to promote clotting and wound healing, and factors that are necessary to activate platelets or otherwise attract the platelets to, and retain them at, a site of injury.
Both the therapeutic and research uses for platelets require that platelets be available in a form that is biologically active. Currently, platelets for therapeutic uses (e.g., infusion for wound healing) are typically provided as freshly isolated products, which are less than five days old. As can be immediately recognized, maintaining an adequate supply of fresh platelets for use by patients in need is costly and results in loss of a large amount of supplies due to expiration prior to use. Furthermore, because fresh platelets are so important for use in therapy, it is difficult and expensive to obtain those platelets for research purposes. Thus, there is a need in the art for alternatives to fresh platelets for therapy and research.
U.S. Pat. No. 5,622,867 to Livesey et al. discloses a system for cryoprotecting platelets for storage. The system treats fresh platelets with an inhibitor system comprising second messenger effectors. Inhibitors of one or more of the following pathways are added: cAMP, sodium channel, cGMP, cyclooxygenase, lipoxygenase, phospholipase, calcium, proteinase and proteinase, and membrane modification. A cryoprotectant, such as DMSO, maltodextrin, dextran, hydroxyethyl starch, and glucose, is also added where the platelets are to be maintained at low temperatures. Prior to use, the platelets are washed to remove the inhibitors and cryoprotectant.
U.S. Pat. No. 5,656,498 to Iijima et al. discloses freeze-dried platelets and methods of making them. The method comprises pre-treating platelets in blood plasma with a solution containing a saccharide, a biopolymer, an acid, or an acid salt, granulating the treated plasma, rapidly cooling the granules, and freeze-drying the granules.
U.S. Pat. No. 5,736,313 to Spargo et al. discloses freeze-dried platelets and a process for making them. The process of making the freeze-dried platelets comprises pre-incubating the platelets in a phosphate-citrate buffer or a phosphate-phosphate-citrate buffer, both of which contain a carbohydrate (e.g., glucose). After pre-incubation, the platelets are loaded with a carbohydrate, then suspended in a lyophilization buffer containing a matrix-forming polymer and a carbohydrate. The platelets are then slowly cooled to about −50° C. while the pressure is reduced to a vacuum state.
U.S. Pat. Nos. 5,958,670 and 5,800,978, both to Goodrich et al., also disclose freeze-dried platelets and methods of making them. The inventions disclosed in these patents rely on use of compositions having glass transition temperatures of above about −60° C. The compositions generally comprise a component that is permeable to the platelets (e.g., a carbohydrate, such as a sugar) and a component that is impermeable to the platelets (e.g., gelatin, PEG). To create the freeze-dried platelets, the temperature of the composition is reduced to a point below the glass transition temperature of the composition, and vacuum evaporating or subliming the liquid from the composition. An earlier patent, U.S. Pat. No. 5,213,814, also to Goodrich et al., discloses stabilized platelets and methods of making them. The methods and platelets are suitable for storage of the platelets for extended periods of time at about 4° C. The methods generally comprise immersing platelets in a buffered aqueous solution containing a carbohydrate and a biologically compatible polymer or mixture of polymers, then freezing the solution and drying the frozen solution to produce freeze-dried platelets containing less than 10% by weight of moisture.
U.S. Pat. Nos. 6,127,111 and 6,372,423, both to Braun, disclose freeze-dried platelets and methods of making them. The methods of making the freeze-dried platelets comprise exposing the platelets to a coagulation inhibitor (e.g., EDTA or citrate) and a “cake forming agent” (e.g., a protein such as serum albumin, or a polysaccharide such as mannitol) for about 5 to 60 minutes at room temperature, then freeze-dried to reduce the moisture content to below 10%.
Investigators at the University of California, Davis, have developed a process for making freeze-dried platelets. The process comprises loading the platelets with trehalose prior to freeze-drying. In U.S. Pat. No. 6,723,497, a method of preparing freeze-dried platelets is disclosed in which platelets are loaded with trehalose by incubating the platelets at a temperature from about 25° C. to less than about 40° C. with up to 50 mM trehalose, cooling the loaded platelets to below −32° C., and lyophilizing the cooled platelets. Published U.S. patent application 2005/0048460 discloses a method for making freeze-dried platelets that includes exposing the platelets to a carbohydrate (e.g., trehalose) and an amphiphilic agent (e.g., arbutin), and freeze-drying the platelets. See, for example, U.S. Pat. Nos. 6,770,478, 6,723,497, 5,827,741, and U.S. published patent applications Nos. 2005/0048460, 2004/0152964, 2004/0147024, and 2004/0136974.
U.S. Pat. No. 6,833,236 to Stienstra discloses a method for the production of stabilized platelets, and platelets made by the method. The method comprises pre-activating the platelets, for example by exposing them to stress, to induce formation of microvesicles, contacting the pre-activated platelets with a carbohydrate to introduce the carbohydrate into the platelets, and drying the loaded platelets.
Even though numerous advances in blood products and wound healing have taken place over the last several years, there is still a need for improved compositions for treating wounds, such as by hemostasis or clotting of wounds. There is accordingly a need for improved methods of making compositions for treating wounds. Likewise, there is a need for methods of treating wounds to stop blood loss that are rapid, effective, and suitable for use in multiple settings. Furthermore, there is still a need for improved diagnostic assays for bleeding diseases and disorders.